Cell Signaling
1. (12 pts) Remember the giant squid recorded Alice’s Scientific Journal? These LGW (Looking Glass
World) predators have complex muscular and nervous systems, and their excitatory cells (and
surrounding fluids) exhibit the ionic concentrations (in mM) listed in the Table below. Trans Looking
Glass monovalent ions differ from ours, and the ones commonly found in organisms on the other side
are represented in the Table below:
N+
OIon:
M+
Inside cells 260
4
34
Outside cells 10
290
300
Patch-clamping measurements indicate stimulated squid nerve and muscle cells exhibit striking
changes in membrane potential as indicated below; these changes are called LAPs (or Looking-Glass
Action Potentials). The resting membrane potential is +88 mV, and the log values for the ratio of the
inorganic ions are indicated along the side. Alice found the temperature over there a uniform
temperature of 298 o K
Logs of Concentration Ratios:
[M+]in/[M+]out = 1.41
[M+]out/[M+]in = -1.41
[N+]in/[N+]out = -1.86
[N+]out/[N+]in: = 1.86
[O-]in/[O-]out = -0.95
[O-]out/[O-]in = 0.95
Answer all the following
questions, showing all
relevant calculations.
A. (4 pts) How is the resting potential likely generated in these cells? Briefly explain the basis for
your answer.
B. (4 pts) Describe the LAP and what changes in membrane properties likely produce its various
features.
C. (4 pts) LAPs may be stimulated in the lab by decreasing the resting potential electronically. How is
a LAP likely stimulated in situ when, for example, a squid motor nerve stimulates a muscle fiber?
CS - 1
Cell Signaling
2. (22 pts) Tetanus, or extreme muscle rigidity, refers to both a normal and a pathological
condition. Normally, a skeletal muscle fiber may be kept in a fully contracted state for a
brief period of time, by the rapid "firing" of the nerve enervating that fiber: thus, during
tetanic stimulation, a high frequency of action potentials (AP's) travel down the nerve to its
synapse with the muscle fiber and result in constant contraction. Widespread, unregulated
tetanus can also be produced in many muscles within an individual by the toxic secretion of
the bacterium, Clostridium tetani, growing anaerobically in a sealed wound.
A. (6 pts) How might the high frequency of nerve AP's continually stimulate fiber contraction?
Briefly describe our present knowledge of the intervening steps, using diagrams as
appropriate.
B. (10 pts) Clinical studies indicate tetanus is produced by very low concentrations of C. tetani
toxin, a large hydrophilic protein. How might the toxin work in such small concentrations?
Propose two different mechanisms based on your knowledge of muscle contraction and its
neural regulation and other aspects of cell signaling.
C. (6 pts) Describe briefly and concretely a test of one of your hypotheses and indicate clearly
what the results would show.
CS - 2
Cell Signaling
3. (15 pts) Gorgonian corals (the sea fans and their relatives) are not preyed upon as heavily
as many of their smaller cousins. Recently, scientists have isolated a lethal neurotoxin,
lophotoxin (LTX), from these creatures. When administered to experimental animals, the first
symptom observed was ataxia (loss of muscular coordination), which was followed quickly by
general paralysis, curtailment of breathing and death. In isolated nerve + muscle
preparations, LTX in very low concentrations inhibited neural stimulated contraction without
affecting the muscle's ability to respond to direct electrical stimulation.
A. (10 pts) Postulate a mechanism for lophotoxin's effect that would account for these
observations.
B. (5 pts) Describe one test of your hypothesis and indicate clearly the expected results.
CS - 3
Cell Signaling
4. (16 pts) I keep a Venus Fly Trap on the kitchen window sill at home to eliminate cluster
flies. When a fly bends two or more sensory "hairs" on the inside surface of the trap, or the
same hair two or more times in quick succession, the trap closes fairly rapidly and finis to the
fly! The inside surface of these traps is moistened with a fluid low in Na+, K+ and Cl-, and the
sensory hairs consist of a complex tissue column of cells. Stuart Jaconson (1965) measured
the plasma membrane potential of these hair cells and found that when bent they exhibited
small, but prolonged, changes in membrane potential, as exhibited below in two
representative traces.
A. (10 points) Ignoring the shape variations explain as concretely as you can how these
changes in membrane potential are likely generated.
B (6 pts) It seems obvious the trap is closing in response to the bending of the hair cells and
the changes in membrane potential, but how might you prove a causal relationship between
these events? Briefly describe an experiment and the expected results.
CS - 4
Cell Signaling
5. (24 pts) Members of the giant algal genus, Nitella, exhibit a transmembrane potential of
approximately -140 mV. If stimulated in an appropriate manner - electrically or by bending
the long, narrow cell - the membrane potential changes as indicated in the figure below
(where the arrow indicates the stimulus application). The ambient temperature is 18 oC
The organism is commonly found in fresh-water ponds and streams, and one determination of
the cytoplasmic and environmental concentration of the major ions yielded the following data:
Compartment
Na+
K+
Cl-
Cytoplasm
14.0 mM
119.0 mM
65.0 mM
Stream
1.0
0.1
1.2
Given your understanding of membrane potentials in other cells, answer all the following
questions. (Note: the log of 1/14 is -1.15, of 14/1= 1.15; of 0.1/119 is -3.08, of 119/0.1 = 3.08;
and of 1.2/65 is -1.7. of 65/1.2 = 1.7.)
A. (6 pts) how might these ion gradients be established and maintained?
CS - 5
Cell Signaling
B. (6 pts) How might the "resting" membrane potential arise?
C. (6 pts) How might the "action" potential be generated?
D. (6 pts) Propose a test for one of your hypotheses (in A., B. or C.) and indicate clearly what
the results would show.
CS - 6
Cell Signaling
6. (15 pts) Physiological work using patch-clamp techniques during the past decade has firmly
established the existence of K+ channels in the plasma membranes of plant cells. Given the
appearance of "action" potentials in plant cells (as exhibited in the previous two questions),
how would you expect the molecular properties of these channels to compare with their animal
cell counterparts? Be specific and use diagrams to illustrate your discussion.
CS - 7
Cell Signaling
7.(30 pts) According to your textbook, a mitogen is "a soluble substance, usually a protein, that
induces mitosis in a resting population of cells, thereby causing the cells to resume
proliferation." We haven't studied mitogens, per se, and you should answer the following
questions extrapolating from material you have learned this term.
A. (12 pts) Present a hypothetical scheme to account for the action of a mitogen on cell
proliferation, beginning from the effect of increased mitogen concentration in the extracellular
space and ending with mitosis, and using appropriate diagrams
CS - 8
Cell Signaling
B. (6 pts) Choose one aspect of your scheme - the initial step or the nuclear events, for
example - and describe one test of its validity, indicating clearly what the results of the test
would show.
C. (6 pts) Scientists have suggested some forms of cancer may be the result of mitogen activity.
Describe one genetic mechanism whereby normal growth under control of a mitogen might
become carcinogenic.
D. (6 pts) Assuming you wished to develop a safe, specific drug that would interfere with
the mitogen or otherwise inhibit its effect, towards what part of your scheme would you target
the drug? Why?
CS - 9
Cell Signaling
Video Problem - Fertilization: the First Minute
[Scroll Bar]
8. The video in this problem presents the first 69 sec of a sea urchin's life as viewed
simultaneously with phase contrast and fluorescence microscopy, speeded up about 8-fold. To
fully appreciate fertilization you should first identify the important features of the egg present
before fertilization (and the video) begins and after the video has ended, and imagine how any
changes you observe might be caused and, in turn, related to the process.
The two images on the left below represent the same unfertilized sea urchin egg viewed
simultaneously with phase contrast and fluorescence optics. The egg is approximately 105
mm in diameter, has had its enveloping jelly coats removed and has been injected with Ca
green dextran (a dye which fluoresces when it binds with calcium ions). It is surrounded by
sperm, most of which are out-of-focus. Identify the designated features in the unfertilized egg
and note its fluorescence image is uniformly dark, indicating little or no calcium ions are
present at the µM level. The labeled arrows indicate of the left and right, respectively, which
sperm actually fertilizes the egg and where fluorescence begins to increase following
fertilization. Double-click on the image to check your identifications and then turn the page
[hyperlink to <fertafternolab.gif> in left frame below] to examine the same egg a little over a
minute later, at the end of the video sequence. Note in particular the elevated structure
surrounding the fertilized egg and the uniformly and faintly fluorescing cytoplasm. Again
check your answers and turn the page a second time to bring up the video screen.
Run the video several times, alternately watching the phase contrast and fluorescence images.
Once you come to appreciate the over-all process, replay the video from the beginning one
frame at a time and note the occurrence of two fluorescence events: one early on and both very
rapid and very brief; the other begins later and is more gradual and long-lasting. The first 9
frames of the video are separated by 0.5 sec intervals; the final 43 frames were captured 1.5
sec apart.
Questions
[scroll bar]
For all its specialized functions, an egg is still a
cell and should reasonably exhibit many features
found in other cells. Consider crtically the
accuracy of this expectation and specifically
what you know about the important roles carried
out by calcium in other cells, examine the still
images, video and read the accompanying
caption, and answer the following questions.
1. Briefly describe the cytological events that
accompany sea urchin fertilization.
2. Describe the changes in fluorescence that
accompany fertilization – both the "fast" and the
"slow" - and how each fluorescent wave changes
with time. (Note the "fast" occurs during the
half-sec between frame 3 and frame 4.)
hyperlink to <fertbeforelab.gif>
CS - 10
Cell Signaling
3. If both fluorescence events represent changes
in the calcium concentration of the egg, what
might be the origin of the calcium in each
instance?
4. How might you test your hypothesis?
5. How might sperm contact with the egg initiate
each of these fluorescence events?
6. What causes the elevation of the fertilization
envelope, and what role, if any, might calcium
play in this process? First answer this question,
and then turn the page <hyperlink to CortGran>
to consider the effect of calcium on the cortical
granules, which are organelles found in the egg
cortex.
“CortGran” – Caption/upper frame
The Egg Cortex
[scroll bar]
To understand the egg’s response to sperm contact, let’s focus on the egg cortex, a region that
lies just beneath the plasma membrane, as illustrated in the figure above. The cortex is filled
with many small vesicles of uniform size called, not surprisingly, cortical granules; these lie
adjacent to and almost touching the plasma membrane.
Once you become familiar with this cortical view, turn to the next Figure to examine the same
field of view with two difference sets of optics. On the right, differential interference contrast
microscopy produces an “optical slice” of a layer of cortical granules just beneath the plasma
membrane; on the left, a fluorescence microscope illustrates the same optical slice. The egg is
suspended in sea water containing a water-soluble dye that becomes fluorescent in a lipid
environment (but which is non-fluorescent in an aqueous one). In this initial frame none of
the granules fluoresce, although the overlying plasma membrane (which is out of the plane of
focus) is fluorescent.
As the video runs in the next frame, watch the behavior of the cortical granules, especially
those enclosed by green circles: the frame interval is 0.5 sec. Try stepping the video one frame
at a time, to characterize the behavior of these four granules, and then consider the questions
in the side-bar.
[CortGran – lower frame]
CS - 11
Cell Signaling
<hyperlink with following split frame>
[Lower frame]
Questions
1. Describe carefully what happens to the 4
granules, correlating the DIC and fluorescent
images.
2. How might the two images be causally
related: i.e., how might the change in the lefthand image produce the change in the righthand image?
3. How might you test your hypothesis.
[hyperlink with <terasaki3.cortgran.mov>
[You may obtain more information about signaling and cortical granules mechanisms in sea
urchin fertilization in Terasaki, M. 1998. Mol. Biol. Cell 9:1609-1612. PMID:9658156
(PubMed ID number)]
9. Pictured below are typical cartoons of two monovalent cation channels that are found in
the plasma membrane of different nerve and muscle cells.
CS - 12
Cell Signaling
One can imagine a variety of questions being asked about these channels (at different levels of
difficulty); here are some examples: (Can you think of others?)
A. Being integral membrane proteins, these channels are very hydrophobic molecules and not
easily assessed by most biochemical techniques. How then were the structures depicted in the
figures determined?
CS - 13
Cell Signaling
B. How do these channels function? Draw their likely three-dimensional structures and
briefly describe, with appropriate labels, how the various domains and regions are thought to
operate .
C. Which cartoon represents the Na+ channel, the K+ channel?
CS - 14
Cell Signaling
D. Relatively speaking, K+ channels are highly diversified in terms of structure and function,
while Na+ channels are a more close knit family. Many cells, for example, exhibit one Na+
channel and several different K+ channels, and the variation in the latter may be even greater
when different tissues are examined. Briefly describe at least two different types of K+
channels and speculate how such variety might be generated mechanistically and why it
might have arisen in the course of evolution.
CS - 15